PROJECT SUMMARY/ABSTRACT: The apical membranes of the retinal pigment epithelium (RPE) are associated with the photoreceptor outer segments (POS), the light-sensing organelle of the neurons. POS contain a large amount of the visual pigments consisting of an 11-cis-retinal (11cRAL), the light-sensitive chromophore, and an opsin G protein-coupled receptor. When light hits the visual pigments, photon energy activates opsins by converting 11cRAL to all-trans- retinal (atRAL) in the visual pigments. Since apo-opsins (without 11cRAL) do not respond to light stimulation, 11cRAL must be regenerated and recombined with apo-opsins to form light sensitive visual pigments. It is well established that atRAL produced by the photoisomerization is reduced to all-trans-retinol (atROL) in the POS; atROL is then released from the POS into the interphotoreceptor matrix (IPM) between POS and the RPE apical membranes, and esterified to all-trans retinyl esters (atRE) in RPE in order to synthesize 11cRAL. However, it is largely unknown how RPE uptakes atROL from the IPM and how the esterification of atROL in RPE is regulated. Interphotoreceptor retinoid-binding protein (IRBP) secreted from the photoreceptors is the most abundant soluble protein in the IPM. Mutations in IRBP cause vision impairment and retinal dystrophy in affected patients and Irbp-/- mice. We recently demonstrated that IRBP plays an important role in the retinoid visual cycle. Consistent with these studies, our preliminary studies showed that IRBP promoted intracellular atRE synthesis in the eyecup RPE incubated with the extracellular atROL in the presence of IRBP. This result suggests that IRBP facilitates atROL uptake and/or esterification by RPE. However, the molecular mechanism by which IRBP promotes cellular uptake and esterification of atROL remains completely unknown. The goal of this project is to identify and characterize RPE apical membrane and intracellular proteins involved in the IRBP-dependent atROL uptake and atRE synthesis by RPE. Through screening of bovine RPE cDNA libraries, we have isolated a gene for an intracellular apical protein that promoted atRE synthesis from the extracellular substrate of atROL bound with IRBP. Aim 1 is to elucidate the molecular mechanism by which the apical protein promotes atRE synthesis in RPE. To do this, we will analyze interaction of the protein with cellular retinol-binding protein 1 (CRBP) and/or lecithin:retinol acyltransferase (LRAT) in RPE. Aim 2 is to characterize an apical transmembrane protein as a potential receptor for IRBP. Our preliminary study showed that an RPE apical transmembrane protein interacted with IRBP. We will test if the interaction promotes synthesis of atRE in RPE. We will then test if eliminating the membrane protein in the mouse RPE reduces atRE synthesis. The results of this project will discover previously unknown players involved in the IRBP-dependent atROL uptake and esterification, establishing a novel regulatory mechanism of the visual cycle. The results may also provide a knowledge basis for the development of a new therapy alleviating vision impairment and retinal degeneration in patients with IRBP mutations.